Cytokinins: Biosynthesis metabolism and perception

Summary Cytokinins are essential hormones for plant growth and development. They are also of vital importance for in vitro manipulations of plant cells and tissues. The biological activities and chemistry of cytokinins are well defined but very little is known about their mode of action and it is only recently that cytokinin genes have been identified in plants. This review summarizes the current status of knowledge on cytokinin biosynthesis, metabolism and signal transduction, with an emphasis on genes encoding metabolic enzymes and putative receptors, and genes rapidly induced by cytokinins.     

Comparison of effects of bacterial strains differing in their ability to synthesize cytokinins on growth and cytokinin content in wheat plants

The content of cytokinins and pigments together with the morphological parameters and fresh weight were estimated in durum wheat (Triticum durum Desf.) plants 2–4 days after introduction into their rhizosphere of an aliquot of Bacillus suspension using the strains that differed in their ability of producing cytokinins. The experiments were performed under laboratory conditions at the optimum light intensity and mineral nutrition. Inoculation with microorganisms incapable to synthesize cytokinins did not affect the total cytokinin content in the wheat plants, whereas the presence of cytokinin-producing microorganisms in the rhizosphere was accompanied by a considerable increase in the total cytokinin content and the accumulation of individual hormones. On the second day after inoculation, a dramatic increase in zeatin riboside and zeatin O-glucoside contents was observed in the roots, and at the next day the accumulation of zeatin riboside and zeatin was registered in the shoots of treated plants. The increase in cytokinin content promoted plant growth (the increased leaf length and width and a faster accumulation of plant fresh and dry weight). Plant treatment with a substance obtained from microorganisms incapable to synthesize hormones resulted in the insignificant growth stimulation. Plant treatment with a substance obtained from cytokinin-producing microorganisms increased leaf chlorophyll content; in this case, the level of chlorophylls was comparable to that observed in the plants treated with a synthetic cytokinin benzyladenine. The role of cytokinins of microbial origin as a factor providing for growth-stimulating effect of bacteria on plants is discussed.     

Molecular Cloning and Characterization of a Cytokinin Dehydrogenase Gene from Upland Cotton (Gossypium hirsutum L.)

Cytokinins are plant hormones that play crucial roles in plant growth and development. Cytokinin dehydrogenase (CKX), regarded as a main negative regulator in cytokinin metabolism in plants, irreversibly degrades cytokinins into adenine/adenosine moiety. A CKX homologous gene, designated GhCKX, was cloned from upland cotton (Gossypium hirsutum L.). Transgenic tobacco plants over-expressing GhCKX showed a typical cytokinin-deficient phenotype, while CKX-silenced tobacco plants exhibited cytokinin over-producing phenotype. Tissue specifically enhancing the expression of GhCKX in the ovule epidermis of transgenic cotton led to a significant decrease of trans-zeatin and trans-zeatin riboside contents in the ovule. The decline of cytokinins resulted in a significant decrease in fiber initials on a single ovule. Our results indicate that GhCKX encodes a functional CKX, and cytokinins may be required for the initiation of cotton fiber cells.     

Cytokinin biosynthesis and transport for systemic nitrogen signaling

The plasticity of growth and development in response to environmental changes is one of the essential aspects of plant behavior. Cytokinins play an important role as signaling molecules in the long-distance communication between organs in systemic growth regulation in response to nitrogen. The spatial distribution of the expression sites of cytokinin biosynthesis genes leads to structural differences in the molecular species transported through the xylem and phloem, giving root-borne trans-hydroxylated cytokinins, namely trans-zeatin (tZ) type, a specialized efficacy in regulating shoot growth. Furthermore, root-to-shoot translocation via the xylem, tZ, and its precursor, the tZ riboside, controls different sets of shoot growth traits to fine-tune shoot growth in response to nitrogen availability. In addition to nitrogen, photosynthetically generated sugars positively regulate de novo cytokinin biosynthesis in the roots, and contribute to plant growth under elevated CO₂ conditions. In shoot-to-root signaling, cytokinins also play a role in the regulation of nutrient acquisition and root system growth in cooperation with other types of signaling molecules, such as C-TERMINALLY ENCODED PEPTIDE DOWNSTREAMs. As cytokinin is a key regulator for the maintenance of shoot apical meristem, deepening our understanding of the regulatory mechanisms of cytokinin biosynthesis and transport in response to nitrogen is important not only for basic comprehension of plant growth, but also to ensure the stability of agricultural production.     

Cytokinins and plant immunity: old foes or new friends?

Cytokinins are plant growth promoting hormones involved in the specification of embryonic cells, maintenance of meristematic cells, shoot formation and development of vasculature. Cytokinins have also emerged as a major factor in plant-microbe interactions during nodule organogenesis and pathogenesis. Microbe-originated cytokinins confer abnormal hypersensitivity of cytokinins to plants, augmenting the sink activity of infected regions. However, recent findings have shed light on a distinct role of cytokinins in plant immune responses. Plant-borne cytokinins systemically induce resistance against pathogen infection. This resistance is orchestrated by endogenous cytokinin and salicylic acid signaling. Here, we discuss how plant- and pathogen-derived cytokinins inversely affect the plant defense response. In addition, we consider the molecular mechanisms underlying plant-derived cytokinin action in plant immunity.     

Auxin-Cytokinin Interactions in Wild-Type and Transgenic Tobacco

Cytokinins and auxins are important regulators of plant growthand development, but there is incomplete and conflicting evidencethat auxins affect cytokinin metabolism and vice versa. We haveinvestigated these interactions in Nicotiana tabacum L. by separatein planta manipulation of levels of the hormones followed byanalysis of the induced changes in the metabolism of the otherhormone. Cytokinin-overproducing plants (expressing the Agrobacte-riumtumefaciens ipt gene) had lower than wild-type levels of freeIAA, and reduced rates of IAA synthesis and turnover, but therewere no differences in the profiles of metabolites they producedfrom fed IAA. Similarly, auxin-overproducing plants (expressingthe A.tumefaciens iaaM and iaaH genes), had lower levels ofthe major cytokinins than wild-type plants and lower cytokininoxidase activity, but there were no differences in the profilesof metabolites they produced from fed cytokinins. The data demonstratethat cytokinin or auxin overproduction decreases the contentof the other hormone, apparently by decreasing its rate of synthesisand/or transport, rather than by increasing rates of turnoveror conjugation. Implications for the importance of cytokinin: auxin ratios in plant development are considered. (Received September 24, 1996; Accepted December 4, 1996)     

Role of cytokinins in stress resistance of plants

The facts of both positive and negative influences of cytokinins on stress resistance of plants are known today. Without pretending to a final choice between these points of view, we have made an attempt to analyze the details of the experiments that gave rise to conclusions about the nature of the effect of cytokinins on the resistance to stress-causing influences with a focus on their intensity and duration. The review deals with the data concerning the influence of different adverse factors on the content of endogenous cytokinins and transduction of cytokinin signals, examines the influence on plant resistance of treatment with exogenous hormone, and the effects of genetic modifications causing changes in cytokinin content and signaling. Resistance is considered not only as a mean of plant survival under severe stress but also as an instrument of maintaining growth rate in plants exposed to moderate stress. Literature data and our own results make it possible to conclude that cytokinins play an important role in formation of plant resistance to adverse influences; however, the effect of these hormones depends on stress intensity. Under moderate stress, cytokinins ensure maintenance of plant growth, whereas a drop in cytokinins hampers growth under a strong influence of adverse factors, which is a prerequisite for mobilization of limited resources characteristic of severe stress and ensures preservation of plant viability.     

细胞分裂素对植物衰老的延缓作用

细胞分裂素是一类重要的植物激素,它可在一定程度上延缓植物的衰老。主要从3个方面综述了细胞分裂素与植物衰老之间的关系,即:(1)植物衰老过程中内源细胞分裂素含量变化;(2)外源细胞分裂素的影响;(3)转入与细胞分裂素的合成、降解相关的基因对植物衰老产生的影响。此外,还从细胞分裂素与糖、与脂质氧化反应以及与其它植物激素的关系方面探讨了细胞分裂素在延缓植物衰老中的作用机理。     

Degradation of cytokinins by cytokinin oxidases in plants

The degradation metabolism of cytokinins is an important process that controls the levels of cytokinin active forms and their distribution in plant tissues. It appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This review attempts to collate the limited information available about this enzyme and introduce new facts, obtained in our laboratory, concerning the mechanism of degradation of cytokinins bearing unsaturated isoprene side chains. However, complete clarification of the effects of cytokinin oxidase on cytokinin regulation and its molecular and biochemical properties will be dependent upon the purification of the protein with cytokinin oxidase activity to homogeneity and progress in the development of requisite molecular probes.     

The interaction of cytokinin with other signals

Cytokinins are important signalling molecules in plants, and recent studies have begun to shed light on the molecular mechanisms underlying their biosynthesis and response pathways. However, from the time of their discovery, it has been clear that cytokinins interact with other signals to regulate plant growth and development. Herein the interaction of cytokinin with three other signals: light, ethylene, and auxin is discussed. The interaction between light and cytokinin signalling, highlighted by recent analysis of cytokinin signalling mutants is reviewed. A discussion of another aspect of cytokinin cross-talk, its induction of ethylene biosynthesis in etiolated Arabidopsis seedlings, and recent studies that have begun to elucidate the mechanism underlying this regulation is also presented. Finally, there is a brief review of the interaction of auxin and cytokinin, and present novel expression profiling data of Arabidopsis seedlings treated with combinations of these two hormones, which provide insight into this interaction.     

Contrasting patterns of cytokinins between years in senescing aspen leaves

Cytokinins are plant hormones that typically block or delay leaf senescence. We profiled 34 different cytokinins/cytokinin metabolites (including precursors, conjugates and degradation products) in leaves of a free‐growing mature aspen (Populus tremula) before and after the initiation of autumnal senescence over three consecutive years. The levels and profiles of individual cytokinin species, or classes/groups, varied greatly between years, despite the fact that the onset of autumn senescence was at the same time each year, and senescence was not associated with depletion of either active or total cytokinin levels. Levels of aromatic cytokinins (topolins) were low and changed little over the autumn period. Diurnal variations and weather‐dependent variations in cytokinin content were relatively limited. We also followed the expression patterns of all aspen genes implicated as having roles in cytokinin metabolism or signalling, but neither the pattern of regulation of any group of genes nor the expression of any particular gene supported the notion that decreased cytokinin signalling could explain the onset of senescence. Based on the results from this tree, we therefore suggest that cytokinin depletion is unlikely to explain the onset of autumn leaf senescence in aspen.     

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.     

Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.     

Cytokinin Profiles of AtCKX2-Overexpressing Potato Plants and the Impact of Altered Cytokinin Homeostasis on Tuberization In Vitro

Genes encoding cytokinin oxidase/dehydrogenase (CKX) enzymes have been used lately to study cytokinin homeostasis in a variety of plant species. In this study AtCKX2-overexpressing potato plants were engineered and grown in vitro as a model system to investigate the effects of altered cytokinin levels on tuber formation and tuber size. Protein extracts from shoots and roots of transformed potato plants exhibited higher CKX activity compared to control plants. Total endogenous cytokinin levels were generally not decreased in AtCKX2 overexpressors. However, levels of bioactive cytokinins were markedly lowered, which was accompanied by increased levels of O- and N-glucosides in some transgenic lines. The AtCKX2-overexpressing plants displayed reduced shoot growth but other symptoms of the ??cytokinin deficiency syndrome?? were not recorded. The transgenic plants were able to produce tubers in noninducing conditions. In inducing conditions they developed larger tubers than control. Tubers were also formed on a greater portion of the analyzed AtCKX2 plants, but with a lower number of tubers per plant compared to control. Taken together, our data suggest that cytokinins cannot be regarded simply as positive or negative regulators of tuberization, at least in vitro. Interactions with other plant hormones that play an important role in control of tuberization, such as gibberellins, should be further studied in detail.     

Regulation of a novel Fusarium cytokinin in Fusarium pseudograminearum

Fusarium pseudograminearum is an agronomically important fungus, which infects many crop plants, including wheat, where it causes Fusarium crown rot. Like many other fungi, the Fusarium genus produces a wide range of secondary metabolites of which only few have been characterized. Recently a novel gene cluster was discovered in F. pseudograminearum, which encodes production of cytokinin-like metabolites collectively named Fusarium cytokinins. They are structurally similar to plant cytokinins and can activate cytokinin signalling in vitro and in planta. Here, the regulation of Fusarium cytokinin production was analysed in vitro. This revealed that, similar to deoxynivalenol (DON) production in Fusarium graminearum, cytokinin production can be induced in vitro by specific nitrogen sources in a pH-dependent manner. DON production was also induced in both F. graminearum and F. pseudograminearum in cytokinin-inducing conditions. In addition, microscopic analyses of wheat seedlings infected with a F. pseudograminearum cytokinin reporter strain showed that the fungus specifically induces its cytokinin production in hyphae, which are in close association with the plant, suggestive of a function of Fusarium cytokinins during infection.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Plant hormone cytokinins control cell cycle progression and plastid replication in apicomplexan parasites

Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites.     

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize

Background and Aims

Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation.

Methods

Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses.

Key Results

During the period 0–28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo.

Conclusions

The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.     

Flow of cytokinins through the environment

Cytokinins are a group of plant growth regulators that elicit a wide range of physiological responses in plants. Cytokinins are not restricted to plants alone but also occur in lower order plants, algae and bacteria as well as in parasitic insects and nematodes. The use of cytokinins, including synthetic cytokinins, in agriculture also contributes to the available cytokinin pool. This review discusses possible ways in which cytokinins move through the environment such as via root exudates, leaf litter and in water systems, thus potentially influencing the growth and development of plants and other organisms. This review highlights the need for a more holistic and ecological approach in order to understand cytokinin regulation and homeostasis in plants.